Shear stress–induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress–induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow

Endothelium-derived microparticles from chronically thromboembolic pulmonary hypertensive patients facilitate endothelial angiogenesis.

11 p.-4 fig.-1 tab.Background: Increased circulating levels of endoglin+ endothelial microparticles (EMPs) have been identified in several cardiovascular disorders, related to severity. Endoglin is an auxilary receptor for transforming growth factor β (TGF-β) important in the regulation of vascular structure.Results: We quantified the number of microparticles in plasma of six patients with chronic thromboembolic pulmonary hypertension (CTEPH) and age- and sex-matched pulmonary embolic (PE) and healthy controls and investigated the role of microparticle endoglin in the regulation of pulmonary endothelial function in vitro. Results show significantly increased levels of endoglin+ EMPs in CTEPH plasma, compared to healthy and disease controls. Co-culture of human pulmonary endothelial cells with CTEPH microparticles increased intracellular levels of endoglin and enhanced TGF-β-induced angiogenesis and Smad1,5,8 phosphorylation in cells, without affecting BMPRII expression. In an in vitro model, we generated endothelium-derived MPs with enforced membrane localization of endoglin. Co-culture of these MPs with endothelial cells increased cellular endoglin content, improved cell survival and stimulated angiogenesis in a manner similar to the effects induced by overexpressed protein.Conclusions: Increased generation of endoglin+ EMPs in CTEPH is likely to represent a protective mechanism supporting endothelial cell survival and angiogenesis, set to counteract the effects of vascular occlusion and endothelial damage.This research was supported by a project grant (PG 11/13/28765) from the British Heart Foundation and by grants from Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to CB)Peer reviewe

The ADMA/DDAH Pathway Regulates VEGF-Mediated Angiogenesis

Objectives— Asymmetrical dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor and cardiovascular risk factor associated with angiogenic disorders. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) promote angiogenesis, but the mechanisms are not clear. We hypothesized that ADMA/DDAH modifies endothelial responses to vascular endothelial growth factor (VEGF) by affecting activity of Rho GTPases, regulators of actin polymerization, and focal adhesion dynamics. Methods and Results— The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo. Conclusion— The ADMA/DDAH pathway regulates VEGF-induced angiogenesis in an NO- and Rac1-dependent manner

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background: Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.Methodology/Principal Findings: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.Conclusions/Significance: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning

Hypoxia perturbs endothelium by re-organizing cellular actin architecture: Nitric oxide offers limited protection

Exposure to hypoxia causes structural changes in the endothelial cell (EC) monolayer that alter its permeability. There was a report earlier of impairment of nitric oxide (NO) production in endothelium. Intervention of NO in the altered cellular arrangements of actin cytoskeleton in endothelium for rectification of paracellular gaps in endothelium under hypoxia was observed. The present study demonstrates hypoxia inducing paracellular gaps in hypoxia exposed blood capillaries in chick embryo extra vascular model. Phalloidin staining confirmed significant polymerization of actin and unique cellular localization of the F-actin bands under hypoxia treatments. Addition of spermine NONOate (SPNO), a NO donor, or reoxygenation to endothelial monolayer attenuated hypoxia-mediated effects on endothelial permeability with partial recovery of endothelial integrity through actin remodeling. The present study indicates link of hypoxia-induced actin-associated cytoskeletal rearrangements and paracellular gaps in the endothelium with a low NO availability in the hypoxia milieu. The author concludes that NO confers protection against hypoxia-mediated cytoskeletal remodeling and endothelial leakiness

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture

In tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture

miR-150-PTPMT1-cardiolipin signaling in pulmonary arterial hypertension.

Circulating levels of endothelial miR-150 are reduced in pulmonary arterial hypertension (PAH) and act as an independent predictor of patient survival, but links between endothelial miR-150 and vascular dysfunction are not well understood. We studied the effects of endothelial miR-150 supplementation and inhibition in PAH mice and cells from patients with idiopathic PAH. The role of selected mediators of miR-150 identified by RNA sequencing was evaluated in vitro and in vivo. Endothelium-targeted miR-150 delivery prevented the disease in Sugen/hypoxia mice, while endothelial knockdown of miR-150 had adverse effects. miR-150 target genes revealed significant associations with PAH pathways, including proliferation, inflammation, and phospholipid signaling, with PTEN-like mitochondrial phosphatase (PTPMT1) most markedly altered. PTPMT1 reduced inflammation and apoptosis and improved mitochondrial function in human pulmonary endothelial cells and blood-derived endothelial colony-forming cells from idiopathic PAH. Beneficial effects of miR-150 in vitro and in vivo were linked with PTPMT1-dependent biosynthesis of mitochondrial phospholipid cardiolipin and reduced expression of pro-apoptotic, pro-inflammatory, and pro-fibrotic genes, including c-MYB, NOTCH3, transforming growth factor β (TGF-β), and Col1a1. In conclusion, we are the first to show that miR-150 supplementation attenuates pulmonary endothelial damage induced by vascular stresses and may be considered as a potential therapeutic strategy in PAH

A New NO-Releasing Nanoformulation for the Treatment of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a chronicand progressive disease which continues to carry an unacceptablyhigh mortality and morbidity. The nitric oxide (NO) pathwayhas been implicated in the pathophysiology and progressionof the disease. Its extremely short half-life and systemiceffects have hampered the clinical use of NO in PAH. In anattempt to circumvent these major limitations, we have developeda new NO-nanomedicine formulation. The formulationwas based on hydrogel-like polymeric composite NO-releasingnanoparticles (NO-RP). The kinetics of NO release fromthe NO-RP showed a peak at about 120 min followed by asustained release for over 8 h. The NO-RP did not affect theviability or inflammation responses of endothelial cells. TheNO-RP produced concentration-dependent relaxations of pulmonaryarteries in mice with PAH induced by hypoxia. Inconclusion, NO-RP drugs could considerably enhance thetherapeutic potential of NO therapy for PAH

Nanoscale Study of Calcium Handling Remodeling in Right Ventricular Cardiomyocytes Following Pulmonary Hypertension

Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca2+ transients along with a higher local Ca2+-release activity. These Ca2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle